Nanodisc-Based Proteomics Identify Caj1 as an Hsp40 with Affinity for Phosphatidic Acid Lipids.

Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking, and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids' poor solubility in aqueous solution. Here, we employ nanodiscs-small, water-soluble patches of a lipid bilayer encircled with amphipathic scaffold proteins-along with quantitative proteomics to identify lipid-binding proteins in Saccharomyces cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that the Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of combining proteomics with lipid nanodiscs to identify and characterize protein-lipid interactions that may not be evident using other methods. Data are available via ProteomeXchange with the identifier PXD027992.

Investigating the stability of the SecA-SecYEG complex during protein translocation across the bacterial membrane.

During posttranslational translocation in Escherichia coli, polypeptide substrates are driven across the membrane through the SecYEG protein-conducting channel using the ATPase SecA, which binds to SecYEG and couples nucleotide hydrolysis to polypeptide movement. Recent studies suggest that SecA is a highly dynamic enzyme, able to repeatedly bind and dissociate from SecYEG during substrate translocation, but other studies indicate that these dynamics, here referred to as "SecA processivity," are not a requirement for transport. We employ a SecA mutant (PrlD23) that associates more tightly to membranes than WT SecA, in addition to a SecA-SecYEG cross-linked complex, to demonstrate that SecA-SecYEG binding and dissociation events are important for efficient transport of the periplasmic protein proPhoA. Strikingly however, we find that transport of the precursor of the outer membrane protein proOmpA does not depend on SecA processivity. By exchanging signal sequence and protein domains of similar size between PhoA and OmpA, we find that SecA processivity is not influenced by the sequence of the protein substrate. In contrast, using an extended proOmpA variant and a truncated derivative of proPhoA, we show that SecA processivity is affected by substrate length. These findings underscore the importance of the dynamic nature of SecA-SecYEG interactions as a function of the preprotein substrate, features that have not yet been reported using other biophysical or in vivo methods.

Negative Stain Single-particle EM of the Maltose Transporter in Nanodiscs Reveals Asymmetric Closure of MalK2 and Catalytic Roles of ATP, MalE, and Maltose.

The Escherichia coli MalE-MalFGK2 complex is one of the best characterized members of the large and ubiquitous family of ATP-binding cassette (ABC) transporters. It is composed of a membrane-spanning heterodimer, MalF-MalG; a homodimeric ATPase, MalK2; and a periplasmic maltose receptor, MalE. Opening and closure of MalK2 is coupled to conformational changes in MalF-MalG and the alternate exposition of the substrate-binding site to either side of the membrane. To further define this alternate access mechanism and the impact of ATP, MalE, and maltose on the conformation of the transporter during the transport cycle, we have reconstituted MalFGK2 in nanodiscs and analyzed its conformations under 10 different biochemical conditions using negative stain single-particle EM. EM map results (at 15-25 Å resolution) indicate that binding of ATP to MalK2 promotes an asymmetric, semi-closed conformation in accordance with the low ATPase activity of MalFGK2 In the presence of MalE, the MalK dimer becomes fully closed, gaining the ability to hydrolyze ATP. In the presence of ADP or maltose, MalE·MalFGK2 remains essentially in a semi-closed symmetric conformation, indicating that release of these ligands is required for the return to the initial state. Taken together, this structural information provides a rationale for the stimulation of MalK ATPase activity by MalE as well as by maltose.

Formation of a Chloride-conducting State in the Maltose ATP-binding Cassette (ABC) Transporter.

ATP-binding cassette transporters use an alternating access mechanism to move substrates across cellular membranes. This mode of transport ensures the selective passage of molecules while preserving membrane impermeability. The crystal structures of MalFGK2, inward- and outward-facing, show that the transporter is sealed against ions and small molecules. It has yet to be determined whether membrane impermeability is maintained when MalFGK2 cycles between these two conformations. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that not only is chloride conductance occurring, but also to a degree large enough to compromise cell viability. Introduction of mutations in the periplasmic gate lead to the formation of a channel that is quasi-permanently open. MalFGK2 must therefore stay away from these ion-conducting conformations to preserve the membrane barrier; otherwise, a few mutations that increase access to the ion-conducting states are enough to convert an ATP-binding cassette transporter into a channel.

TonB-dependent ligand trapping in the BtuB transporter.

TonB-dependent transporters are ß-barrel outer membrane proteins occluded by a plug domain. Upon ligand binding, these transporters extend a periplasmic motif termed the TonB box. The TonB box permits the recruitment of the inner membrane protein complex TonB-ExbB-ExbD, which drives import of ligands in the cell periplasm. It is unknown precisely how the plug domain is moved aside during transport nor have the intermediate states between TonB recruitment and plug domain movement been characterized biochemically. Here we employ nanodiscs, native gel electrophoresis, and scintillation proximity assays to determine the binding kinetics of vitamin B12 to BtuB. The results show that ligand-bound BtuB recruits a monomer of TonB (TonB∆1-31), which in turn increases retention of vitamin B12 within the transporter. The TonB box and the extracellular residue valine 90 that forms part of the vitamin B12 binding site are essential for this event. These results identify a novel step in the TonB-dependent transport process. They show that TonB binding to BtuB trap the ligand, possibly until the ExbB-ExbD complex is activated or recruited to ensure subsequent transport.

Lipopolysaccharides promote binding and unfolding of the antibacterial colicin E3 rRNAse domain.

Nuclease colicins are antibacterial proteins produced by certain strains of E. coli to reduce competition from rival strains. These colicins are generally organized with an N-terminal transport (T)-domain, a central receptor binding (R)-domain, and a C-terminal cytotoxic nuclease domain. These colicins are always produced in complex with an inhibitory immunity protein, which dissociates prior entrance of the cytotoxic domain in the target cell. How exactly colicins traverse the cell envelope is not understood, yet this knowledge is important for the design of new antibacterial therapies. In this report, we find that the cytotoxic rRNAse domain of colicin E3, lacking both T- and R-domains, is sufficient to inhibit cell growth provided the immunity protein Im3 has been removed. Thus, while the T-domain is needed for dissociation of Im3, the rRNAse alone can associate to the cell surface without R-domain. Accordingly, we find a high affinity interaction (Kd ~1-2µM) between the rRNAse domain and lipopolysaccharides (LPS). Furthermore, we show that binding of ColE3 to LPS destabilizes the secondary structure of the toxin, which is expectedly crucial for transport through the narrow pore of the porin OmpF. The effect of LPS on binding and unfolding of ColE3 may be indicative of a broader role of LPS for transport of colicins in general.

Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter.

ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK2 in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK2 is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of Pi limiting, and (iii) the additional presence of maltose stimulates release of Pi, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK2 in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and Pi release. This concerted action explains why ATPase activity of MalFGK2 depends on maltose, and why MalE is essential for transport.

Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation.

The complex MalFGK2 hydrolyzes ATP and alternates between inward- and outward-facing conformations during maltose transport. It has been shown that ATP promotes closure of MalK2 and opening of MalFG toward the periplasm. Yet, why the transporter rests in a conformation facing the cytosol in the absence of nucleotide and how it returns to this state after hydrolysis of ATP is unknown. The membrane domain MalFG may be naturally stable in the inward-facing conformation, or the ABC domain may catalyze the transition. We address this question by analyzing the conformation of MalFG in nanodiscs and in proteoliposomes. We find that MalFG alone exists in an intermediate state until MalK binds and converts the membrane domain to the inward-facing state. We also find that MalK, if overly-bound to MalFG, blocks the transition of the transporter, whereas suppressor mutations that weaken this association restore transport. MalK therefore exploits hydrolysis of ATP to reverse the conformation of MalFG to the inward-facing conformation, a step essential for release of maltose in the cytosol.

FhuA interactions in a detergent-free nanodisc environment.

TonB-dependent membrane receptors from bacteria have been analyzed in detergent-containing solution, an environment that may influence the role of ligand in inducing downstream interactions. We report reconstitution of FhuA into a membrane mimetic: nanodiscs. In contrast to previous results in detergent, we show that binding of TonB to FhuA in nanodiscs depends strongly on ferricrocin. The stoichiometry of interaction is 1:1 and the binding constant KD is ~200nM; an equilibrium affinity that is ten-fold lower than reported in detergent. FhuA in nanodiscs also forms a high-affinity binding site for colicin M (KD ~3.5nM), while ferricrocin renders FhuA refractory to colicin binding. Together, these results demonstrate the importance of the ligand in regulating receptor interactions and the advantages of nanodiscs to study ß-barrel membrane proteins in a membrane-like environment.

Two copies of the SecY channel and acidic lipids are necessary to activate the SecA translocation ATPase.

The SecA ATPase associates with the SecY complex to push preproteins across the bacterial membrane. Because a single SecY is sufficient to create the conducting channel, the function of SecY oligomerization remains unclear. Here, we have analyzed the translocation reaction using nanodiscs. We show that one SecY copy is sufficient to bind SecA and the preprotein, but only the SecY dimer together with acidic lipids supports the activation of the SecA translocation ATPase. In discs, the dimer is predominantly arranged in a back-to-back manner and remains active even if a constituent SecY copy is defective for SecA binding. In membrane vesicles and in intact cells, the coproduction of two inactive SecYs, one for channel gating and the other for SecA binding, recreates a functional translocation unit. These results indisputably argue that the SecY dimer is crucial for the activation of SecA, which is necessary for preprotein transport.

ATP alone triggers the outward facing conformation of the maltose ATP-binding cassette transporter.

The maltose transporter MalFGK(2) is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction.

Phosphatidylglycerol directs binding and inhibitory action of EIIAGlc protein on the maltose transporter.

The signal-transducing protein EIIA(Glc) belongs to the phosphoenolpyruvate carbohydrate phosphotransferase system. In its dephosphorylated state, EIIA(Glc) is a negative regulator for several permeases, including the maltose transporter MalFGK2. How EIIA(Glc) is targeted to the membrane, how it interacts with the transporter, and how it inhibits sugar uptake remain obscure. We show here that acidic phospholipids together with the N-terminal tail of EIIA(Glc) are essential for the high affinity binding of the protein to the transporter. Using protein docking prediction and chemical cross-linking, we demonstrate that EIIA(Glc) binds to the MalK dimer, interacting with both the nucleotide-binding and the C-terminal regulatory domains. Dissection of the ATPase cycle reveals that EIIA(Glc) does not affect the binding of ATP but rather inhibits the capacity of MalK to cleave ATP. We propose a mechanism of maltose transport inhibition by this central amphitropic regulatory protein.

The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.

The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.

Structure-activity analysis of niclosamide reveals potential role for cytoplasmic pH in control of mammalian target of rapamycin complex 1 (mTORC1) signaling.

Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH.

The action of cardiolipin on the bacterial translocon.

Cardiolipin is an ever-present component of the energy-conserving inner membranes of bacteria and mitochondria. Its modulation of the structure and dynamism of the bilayer impacts on the activity of their resident proteins, as a number of studies have shown. Here we analyze the consequences cardiolipin has on the conformation, activity, and localization of the protein translocation machinery. Cardiolipin tightly associates with the SecYEG protein channel complex, whereupon it stabilizes the dimer, creates a high-affinity binding surface for the SecA ATPase, and stimulates ATP hydrolysis. In addition to the effects on the structure and function, the subcellular distribution of the complex is modified by the cardiolipin content of the membrane. Together, the results provide rare and comprehensive insights into the action of a phospholipid on an essential transport complex, which appears to be relevant to a broad range of energy-dependent reactions occurring at membranes.

Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome.

Integral membrane proteins are challenging to work with biochemically given their insoluble nature; the nanodisc circumvents the difficulty by stabilizing them in small patches of lipid bilayer. Here, we show that nanodiscs combined with SILAC-based quantitative proteomics can be used to identify the soluble interacting partners of virtually any membrane protein. As a proof of principle, we applied the method to the bacterial SecYEG protein-conducting channel, the maltose transporter MalFGK(2) and the membrane integrase YidC. In contrast to the detergent micelles, which tend to destabilize interactions, the nanodisc was able to capture out of a complex whole cell extract the proteins SecA, Syd, and MalE with a high degree of confidence and specificity. The method was sensitive enough to isolate these interactors as a function of the lipid composition in the disc and the culture conditions. In agreement with a previous photo-cross linking analysis, YidC did not show any high-affinity interactions with cytosolic or periplasmic proteins. These three examples illustrate the utility of nanoscale lipid bilayers to identify the soluble peripheral partners of proteins intergrated in the lipid bilayer.

The SecY complex forms a channel capable of ionic discrimination.

Protein translocation across the bacterial membrane occurs at the SecY complex or channel. The resting SecY channel is impermeable to small molecules owing to a plug domain that creates a seal. Here, we report that a channel loosely sealed, or with a plug locked open, does not, however, lead to general membrane permeability. Instead, strong selectivity towards small monovalent anions, especially chloride, is observed. Mutations in the pore ring-structure increase both the translocation activity of the channel and its ionic conductance, however the selectivity is maintained. The same ionic specificity also occurs at the onset of protein translocation and across the archaeal SecY complex. Thus, the ion-conducting characteristic of the channel seems to be conserved as a normal consequence of protein translocation. We propose that the pore ring-structure forms a selectivity filter, allowing cells to tolerate channels with imperfect plugs.

Selectively targeting the dimerization interface of human androgen receptor with small-molecules to treat castration-resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of death for men in North America. The androgen receptor (AR) - a hormone inducible transcription factor - drives expression of tumor promoting genes and represents an important therapeutic target in PCa. The AR is activated by steroid recruitment to its ligand binding domain (LBD), followed by receptor nuclear translocation and dimerization via the DNA binding domain (DBD). Clinically used small molecules interfere with steroid recruitment and prevent AR-driven tumor growth, but are rendered ineffective by emergence of LBD mutations or expression of constitutively active variants, such as ARV7, that lack the LBD. Both drug-resistance mechanisms confound treatment of this 'castration resistant' stage of PCa (CRPC), characterized by return of AR signalling. Here, we employ computer-aided drug-design to develop small molecules that block the AR-DBD dimerization interface, an attractive target given its role in AR activation and independence from the LBD. Virtual screening on the AR-DBD structure led to development of prototypical compounds that block AR dimerization, inhibiting AR-transcriptional activity through a LBD-independent mechanism. Such inhibitors may potentially circumvent AR-dependent resistance mechanisms and directly target CRPC tumor growth.